1. Field of the Invention
This invention concerns super-paramagnetic particles with an increased R1 relaxivity which can be used as diagnostics in NMR tomography and as a pharmacologically active substance. This invention also concerns a production process and the use of the new particles.
2. The Prior Art
European Patent No B 284,549 describes super-paramagnetic single-domain particles of iron oxide, mixed iron oxides or iron with a particle size in the range between 3 and 20 nanometers, having organic substances of the group consisting of polyalkylene glycols containing phosphate groups, diphosphate groups, polyphosphate groups, thiophosphate groups, phosphonate groups or thiophosphonate groups, nucleotides containing phosphate groups, their oligomers or polymers and phosphate group-containing carbohydrates chemically bound at their surface, optionally having additional binding sites.
German Patent No. DE-A-4,309,333 describes stable, degradable aggregates with a particle size in the range between 10 and 1000 nanometers with defined behaviour in the magnetic field, with the aggregates consisting of multiple small super-paramagnetic single-domain particles of iron oxide, mixed iron oxides or iron with a particle size in the range between 3 and 20 nanometers, having substances of the group of polyalkylene glycols containing phosphate groups, diphosphate groups, polyphosphate groups, thiophosphate groups, phosphonate groups or thiophosphonate groups, or having carbohydrates or phosphate group-containing nucleotides their oligomers or polymers chemically bound at their surface.
WO 96/03653 describes super-paramagnetic particles consisting of super-paramagnetic single-domain particles and aggregates of super-paramagnetic single-domain particles having organic substances bound at their surface. The super-paramagnetic particles are composed of a mixture of small super-paramagnetic single-domain particles with a particle size in the range between 3 and 50 nanometers and stable, degradable aggregates of small super-paramagnetic single-domain particles with a particle size in the range between 10 and 1000 nanometers and consist of iron hydroxide, iron oxide hydrate, iron oxide mixed iron oxides or iron having mono- and/or polyhydroxyl group-containing aromatic substances, polyglycerols, amino acid-containing substances, silicate group-containing substances of ortho-silicic acid and their condensation products and phosphate group-containing substances of ortho- or meta-phosphoric acid and their condensation products bound at their surface.
Super-paramagnetic single-domain particles with a particle size in the range between 3 and 50 nanometers can be produced according to the state of the art and used, for example, as contrast media for NMR tomography, with preferred particle diameters in the range of 5-20 nm. In the regions of the body where these particles have accumulated, they lead to a greater shortening of the 2-relaxation time, and to darkening of the signal. Therefore, one also speaks of xe2x80x9cnegativexe2x80x9d NMR contrast media with super-paramagnetic single-domain particles. The T relaxation time is inversely proportional to the relaxivity R, so the relaxivity R increases as the T relaxation time is shortened. The T2 relaxation time has a linear dependence on the particle diameter of the magnetic particles. The larger the particle diameter, the greater the shortening of the T2 relaxation time and the lower the required particle concentration to achieve a xe2x80x9cnegativexe2x80x9d contrast. The contrast medium demand is in the range of 10 to 20 mmol Fe/kg body mass, and thus is much lower in comparison with the xe2x80x9cpositivexe2x80x9d NMR contract media based on gadolinium chelates used in the past.
The T1 relaxation time of such xe2x80x9cnegativexe2x80x9d contrast media is not shortened greatly, so that there is no mentionable signal amplification in the NMR tomography in the body regions where they have accumulated. There is little lightening of the signal in the tissue. For many applications, it would be advantageous to be able to use super-paramagnetic single-domain particles with an increased xe2x80x9cpositivexe2x80x9d NMR contrast medium effect.
If T1-weighted NMR tomography is to be performed, then the ratio of the relaxivities R2/R1 must be as small as possible, but at least smaller than 5.
The R1 relaxivity cannot be changed much with a reduction in particle diameter, but the R2 relaxivity can be reduced. With very small super-paramagnetic single-domain particles, the ratio of relaxivities R2/R1 is reduced so greatly that T1-weighted NMR tomography can be performed. Since super-paramagnetic single-domain particles have an influence on signal intensity even in very low concentrations, they can be superior to the paramagnetic contrast media used in the past.
The object of the present invention is to develop very small super-paramagnetic single-domain particles which can be used as contrast media for T1-weighted NMR tomography, for example.
According to this invention, this can be achieved by super-paramagnetic single-domain particles with an increased R1 relaxivity and with surface stabilizer substances, comprising particles of iron hydroxide, iron oxide hydrate, iron oxide, mixed iron oxides or iron with a particle size in the range between one and ten nanometers, with an average particle diameter d50 of two to four nanometers, an increased R1 relaxivity in the range of two to fifty, an R2/R1 relaxivity ratio of less than five, and having stabilizer substances on their surface of aliphatic dicarboxylic acids, aliphatic polycarboxylic acids substitution products thereof and derivatives thereof. That stabilizer substances prevent aggregation and sedimentation of the particles in a gravitational field or a magnetic field. On the stabilized particles are optionally bound further stabilizer substances, tissue-specific binding substances, pharmacologically active substances, pharmacologically active cells, pharmacologically active chelating agents, cell fusion mediating substances or gene transfer mediating substances, as well as mixtures thereof.
The super-paramagnetic single-domain particles of the invention with an increased R1 relaxivity have on its surface first stabilizer substances.
If the first stabilizing substances are aliphatic dicarboxylic acids they are preferably malic acid, tartaric acid, glucaric acid.
If the first stabilizing substances are aliphatic polycarboxylic acids they are preferably citric acid, cyclohexanetricarboxlic acid, cyclohexanehexacarboxylic acid, ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid.
Substitution products of dicarboxylic or polycarboxylic acids are compounds with unchanged carboxyl groups but one or more replaced hydrogen atoms by alkyl groups, halogen atoms or other groups such as citramalic acid, 2-methylene malic acid, alpha-hydroxycitric acid, aspartic acid, glutamic acid, pteroglutamic acid (folic acid).
Derivatives of dicarboxylic or polycarboxylic acids are compounds produced by changing of the carboxyl group such as esters, acid chlorides, anhydrides, amides, hydroxamic acids, imido esters, e.g. preferred C12-C18 fatty acid esters with one or more of the a.m. dicarboxylic or polycarboxylic acids such as citric acid stearyl ester.
Various possibilities of achieving the goal of the inventionxe2x80x94very small superparamagnetic particles for e.g. T1-weighted tomographyxe2x80x94have been found:
1. By reducing the magnetic susceptibility of the super-paramagnetic single-domain particles;
2. By reducing the particle diameter of the super-paramagnetic single-domain particles in the range of 1-10 nm, preferably in the range of 2-4 nm;
3. Through the choice of stabilizer substances, and
4. Through the thickness and the water content of the stabilizer substance layer.
The very small super-paramagnetic single-domain particles may consist of the following substances: iron hydroxide, iron oxide hydrate, xcex3-Fe2O3, Fe3O4, the mixed iron oxides of the general formula m MO.n Fe2O3, where M denotes the divalent metal ions Fe, Co, Ni, Mn, Be, Mg, Ca, Ba, Sr, Cu, Zn, Pt or mixtures thereof, the mixed oxides of the general formula m Fe2O3.n Me2O3, where Me denotes the trivalent metal ions Al, Cr, Bi, rare earth metals or mixtures thereof or iron, and m and n denote integers from 1 to 6. Thus, through the composition and structure of the single-domain particles, their magnetic susceptibility can be varied in a wide range, and an R2/R1 relaxivity ratio of less than 5 can be established. However, it is important for use in medicine to take into account the toxicity of the individual components of the single-domain particles.
The super-paramagnetic particles of the invention are preventing aggregation and sedimentation in a gravitational field or in a magnetic field. That means the aggregation and sedimentation of the particles is prevented in a gravitational field up to 10,000 g for at least 10 minutes, such as during centrifugation with 10,000 rpm (see example 1). The aggregation and sedimentation is prevented also in a magnetic field of about 0.1 Tesla for 30 minutes and for above defined iron/iron oxide particles of 1-10 nm diameter. Said particles of  less than 5 nm are even stable against aggregation and sedimentation in a magnetic field of e.g. about 1 Tesla for 30 minutes. That are usual conditions of an NMR tomography apparatus.
The average particle diameter xe2x80x9cd50xe2x80x9d means that at least 50% of the particles has the diameter 2-4 nm and the other has a diameter  less than 2 nm and between 4 and 10 nm.
The magnetic susceptibility of the very small super-paramagnetic single-domain particles according to the present invention can also be adjusted in a wide range, from approximately 1 to 100 EMU/g, through the composition of the iron salt solution and its method of preparation.
It has surprisingly been found that a change in composition of the iron salt solution has an influence on the R2/R1 relaxivity ratio. The magnetic susceptibility can be reduced from approximately 100 EMU/g to approximately 20 EMU/g by increasing the Fe3+/Fe2+ concentration ratio of the iron salt solution from 2 to 10.
Optionally a change in R2/R1 relaxivity ratio can be achieved by an oxidation or reduction reaction after precipitation of the very small single-domain particles. Suitable redox agents include, for example, nitrate ions, hydrogen peroxide, hydroxylamine, ascorbic acid.
It has surprisingly been found that the particle size of the super-paramagnetic single-domain particles can be reduced even further by precipitating the particles under an elevated temperature and optionally an elevated pressure, with bases from an iron salt solution containing aqueous or water-miscible organic solvents, adding a solution of a described carboxylic acid stabilizer substance in water or water-miscible organic solvent immediately before, during or immediately after precipitation, or by precipitating with a mixture of stabilizer substance and base, or if the stabilizer substance is also a base, by just adding the organic base. A precipitation temperature of 50xc2x0 C. to 120xc2x0 C. can be stipulated as advantageous. At temperatures above 100xc2x0 C., the or is carried out in autoclaves. Adding the stabilizer substance immediately before, during or immediately after precipitation prevents the above-mentioned redox reactions and yields very small super-paramagnetic single-domain particles.
The particle size of the very small super-paramagnetic single-domain particles is in the range of 1 to 10 nm, especially in the range of the average particle diameter d50 of 2 to 4 nm. The stated particle sizes of 1 to 10 nm with an average particle diameter d50 of 2-4 nm always refers to the particles without stabilizer substance.
With a reduction in particle size, the biological tolerability is also improved, and the rate of degradation in the body is increased. The bioavailability of the very small super-paramagnetic particles in the body is a few hours to days, depending on the particle size and the composition of the stabilizer substances, i.e., the reticuloendothelial system binds the very small super-paramagnetic particles relatively slowly.
The object of the present invention is also to expand the range of substances that stabilize the very small super-paramagnetic single-domain particles against aggregation and sedimentation, to optimally adapt the physicochemical and physiological properties of the corresponding magnetic particles to the respective applications, and these substances should be stable and easily synthesized.
It has surprisingly been found that even small molecules, not only the relatively large polymer molecules or macromolecules known in the state of the art, are suitable for stabilization of very small super-paramagnetic single-domain particles.
It has been found that aliphatic di- and polycarboxylic acids and their substitution products and derivatives, such as malic acid, tartaric acid, citric acid, aspartic acid, are suitable stabilizer substances for very small super-paramagnetic single-domain particles.
The very small super-paramagnetic single-domain particles according to this invention, stabilized with citric acid, for example, have much smaller diameters than the smallest super-paramagnetic iron oxides which were provided with a polymer coating and were produced in the past. Thus, for example, it is now possible to produce stabilized super-paramagnetic single-domain particles with an average particle diameter d50 range of 2 to 4 nm that can be filtered through a 100,000 D filter, i.e., they have an average particle diameter of the stabilized particles of 4 to 8 nm. The R2/R1 relaxivity ratios can thus be reduced to values between 1 and 3.
Thus, the half-life of the novel, very small, super-paramagnetic single-domain particles in blood is much longer than that of previous particles, thereby greatly expanding the possible areas of use for T1-weighted NMR tomography e.g. angiography and for diagnosis of thrombi, tumors and inflamed tissues, or for T2-weighted tomography e.g. lymphography and for diagnosis of thrombi, tumors and inflamed tissues, or for T1- and T2-weighted tomography e.g. differentiation diagnostic by use of T1- and T2-weighted images.
It has surprisingly also been found that the type and quantity of stabilizer substances have an influence on the R2/R1 relaxivity ratio of the super-paramagnetic single-domain particles. For example, R2/R1 can be reduced by an increase in the hydrophilic nature of the stabilizer and thus its degree of hydration. The quantity of stabilizer substance also has an influence on particle size. With an increase in the concentration of the stabilizer substance, the particle diameter becomes smaller.
The physicochemical and physiological properties of the resulting very small super-paramagnetic single-domain particles can be optimized for the respective application if additional (further) stabilizer substances are bound at the surface of the particles, i.e. at the first stabilizer substances. These additional stabilizer substances can be selected from aromatic substances containing mono- and polyhydroxyl groups, such as benzenoids, coumarins, lignans, terphenyl, flavonoids, tannins, xanthones, benzophenones, naphthalenes, naphthoquinones, anthraquinones, anthracyclines, polycyclic condensed aromatic compounds and their derivatives; substances containing amino acids, such as albumins, globulins, oligopeptides, polypeptides, denatured products of proteins and proteids, such as gelatins, casein hydrolysate, gluteline; substances containing thio groups, such as mercaptopurine, mercaptocytosine, mercaptoguanine, mercaptouracil, mercaptothymine, mercaptohypoxanthine, and their mercaptonucleosides and mercaptodeoxynucleosides; substances of ortho-silicic acid containing silicate groups and their condensation products with divalent and polyvalent inorganic ions and organic acids, such as phytic acid, alginic acid, gallic acid; substances of ortho- or meta-phosphoric acid containing phosphate groups and their condensation products, such as pyrophosphoric acid, polyphosphoric acids, cyclophosphates and their heterocondensation products, and their reaction products with organic compounds containing basic groups, such as spermine, spermidine, polyethyleneimine, protamines, oxygelatin and their derivatives; organic substances of the group consisting of carbohydrates containing phosphate groups, di-phosphate groups, polyphosphate groups, thiophosphate groups, phosphonate groups, thiophosphonate groups, carboxylate groups, sulfate groups, sulfonate groups, mercapto groups, silanetriol groups; polyalkylene glycols, alkyl, aryl and/or alkylaryl polyethylene glycols; nucleotides that contain phosphate groups, as well as their oligomers or polymers; polysaccharides containing nitrogen, such as mucopolysaccharides, glycoproteids, chitins and their derivatives.
The stabilizer substances can be produced by the state of the art or they can be acquired commercially.
To the first or further stabilizer molecules may be bound all tissue-specific binding substances such as antigens, anti-bodies, ribonucleic acids, deoxyribonucleic acids, ribonucleic acid sequences, deoxyribonucleic acid sequences, haptenes, avidin, streptavidin, protein A, protein G, endotoxin-binding proteins, lectins, selectins, surface proteins of organelles; viruses, microbes, algae, fungi; all pharmacologically active substances such as anti-tumor proteins, enzymes, anti-tumor enzymes, antibiotics, plant alkaloids, alkylating reagents, antimetabolites, hormones and hormone antagonists, interleukins, interferones, growth factors, tumor necrosis factors, endotoxins, lymphotoxins, urokinase, streptokinase, plasminogen streptokinase activator complex, tissue plasminogen activators, desmodus plasminogen activators, macrophage activation bodies, antisera, blood and cell constituents and their degradation products and derivatives, cell wall components of organelles, viruses, microbes, algae, fungi and their degradation products and derivatives, protease inhibitors, alkyl phosphocholines, substances containing radioactive isotopes, surfactants, cardiovascular pharmaceuticals, chemotherapeutics, gastrointestinal pharmaceuticals, neuropharmaceuticals; all pharmacologically active cells such as organelles, viruses, microbes, algae, fungi, especially red blood cells, platelets, granulocytes, monocytes, lymphocytes, islets of Langerhans;
all pharmacologically active chelating agents, such as polycarboxylic acids, polyamino acids, porphyrins, catecholamines;
all substances that promote cell fusion, such as polyethylene glycols, alkyl, aryl and alkylaryl polyethylene glycols and their derivatives;
all substances that mediate gene transfer, such as polyethylene glycol and derivatives thereof; polyamine compounds such as polyethyleneimine, spermine, spermidine, protamine sulfate; as well as mixtures thereof.
There are two possibilities in the state of the art for stabilizing super-paramagnetic particles produced by precipitation reactions against aggregation and sedimentation:
1. The super-paramagnetic particles are prepared by precipitation from the iron salt solutions, purified and then stabilized with the proper stabilizer substance, or
2. The stabilizer substances are mixed with the iron salt solutions and the mixture is heated to the respective precipitation temperature and precipitation is performed.
Since the mixture of Fe3+/Fe2+ salt solution is a reactive redox system, parts of the stabilizer substance may be oxidized or reduced, and the Fe3+/Fe2+ concentration ratio changes in an unreproducible manner. Many possible stabilizer substances or even many organic anions of the iron salt solutions form complexes with the iron salts which also lead to non-magnetic or unreproducible precipitation products.
The method of producing very small super-paramagnetic single-domain particles with an increased R1 relaxivity and with surface stabilizer substances is performed from an iron salt solution at an elevated temperature in the range of 50xc2x0 C. to 120xc2x0 C., with the particles being precipitated with bases, e.g., with alkali lye, ammonia water or organic bases. When the temperatures is greater than 100xc2x0 C., the process is carried out in an autoclave. The precipitation can also be performed with a mixture of stabilizer substance and base, or, if the stabilizer substance is also a base, it can be performed only by adding the organic base. Organic bases, which function as the stabilizer substance at the same time include, for example, polyethyleneimine, spermine, spermidine or protamine. A dissolved stabilizer substance consisting of aliphatic di- and polycarboxylic acids and their substitution products and derivatives such as malic acid, tartaric acid, citric acid, aspartic acid is added to the iron salt solution during precipitation or immediately after precipitation in an amount of 5 to 100 wt %, based on the quantity of single-domain particles.
By adding an oxidizing agent or reducing agent, the Fe3+/Fe2+ concentration ratio in the precipitated product can be adjusted in the range from one to infinite (xcex3-Fe2O3) to achieve the desired change in R2/R1 relaxivities. The dispersion of very small, stabilized super-paramagnetic particles prepared in this way is cooled and neutralized with hydrochloric acid, for example, the dispersion is dialyzed until the electric conductivity of the filtrate is  less than 10 mS/cm.
With some stabilizer substances, it is necessary to input more energy, e.g., by the action of ultrasound, to prepare stable dispersions. The stable dispersion may also contain larger or weakly aggregated super-paramagnetic single-domain particles, which can easily be separated from the very small super-paramagnetic particles by sedimenting them in a magnetic field or by centrifugation. Optionally additional substances of the group of further stabilizer substances, tissue-specific binding substances, pharmacologically active substances, pharmacologically active cells, pharmacologically active chelating agents, cell fusion mediating substances and gene transfer mediating substances, as well as mixtures thereof to which the very small stabilized super-paramagnetic single-domain particles are coupled.
Tissue-specific binding substances are substances such as rutin which in a mixture with e.g. citric acid is bound to the surface of the superparamagnetic particles as a new stabilized preparation having very good binding characteristics to the cell wall parts of e.g. tumor cells. It has been found that after injection of such a preparation on rats the tumor growth reduced half the time (see example 9).
Further it has been found that superparamagnetic particles of the present invention stabilized with an aliphatic carboxylic acid such as citric acid are added on neutrophilic white blood cells as a representative of the group of pharmacologic active cells. The xe2x80x9cmagnetizablexe2x80x9d white blood cells enriches also in inflamed tissue and can be used as contrast medium in NMR for the recognition of inflamed tissue. These xe2x80x9cmagnetizablexe2x80x9d white blood cells enriches also on tumor edges and can be used as contrast medium in NMR for the recognition of tumors. The addition of stabilized superparamagnetic particles on pharmacological active cells takes place ex vivo by mixing the stabilized particles with a blood sample or with a fraction thereof enriched with leucocytes. After an incubation time of about 20 min the injection of the blood sample into the body follows. The NMR contrast effect can be observed after 5-120 min (see ex. 10).
Further it has been found that superparamagnetic particles of the present invention stabilized e.g. with citric acid and tannin bind also on leucocytes. After i.v. injection of a stabilized watery particle dispersion they enrich in the lymphatic node and in the bone marrow. After about 12-24 h the lymphatic node and the bone marrow are visible in a NMR image. Pharmacological effects of the xe2x80x9cmagnetizablexe2x80x9d blood cells are to achieve by heating the strong inhomogenic electromagnetic fields (magnetic field hyperthermie) or by coupling on pharmacologic active substances and/or by radiation with radioactive or particle radiation.
With respect to cell fusion mediating substances and gene transfer mediating substances it is possible to bind on super-paramagnetic particles, stabilized by e.g. an aliphatic carboxylic acid, substances such as methoxy polyethylene glycolphosphate (molecular weight 2000). With such particles cell fusion and gene transfer can be recognized optically by microscoping the cells in the presence of the dark colored magnetic particles, i.e. dark coloring of the cells.
Genes which are to transport by invasion into the cells are mixed with an surplus of superparamagnetic particles stabilized with e.g. an aliphatic carboxylic acid and methoxy polyethylene glycolphosphate (MW 2000) to wrap up the genes with the particles. Not bounded particles are removed by washing with a physiologically acceptable solution. These xe2x80x9cmagnetized genesxe2x80x9d are added to the cell. By microscopy the skilled in the art is able to recognize the dark coloring of the cell content. After i.v. injection of a watery suspension of the xe2x80x9cmagnetized genesxe2x80x9d they enrich in the lymphatic node and in the bone marrow.
Preparation of the very small super-paramagnetic particles according to the present invention is explained here on the basis of examples.